Exopolysaccharides play important roles in determining the virulence of pathogenic bacteria, affecting their adherence to surfaces, and protecting them from antibiotics and the host's immune system. Much remains to be learned about the steps that govern the maturation and transport of exopolysaccharides in important human pathogens. The present application describes investigations into the final steps of alginate biosynthesis in Pseudomonas aeruginosa. Alginate is an unbranched heteropolymeric carbohydrate that forms a capsule around the bacteria. The precursors for polymer synthesis are generated in the cytoplasm, and polymerization to polymannuronan occurs through the action of integral cytoplasmic membrane proteins. We describe experiments to characterize the processes that occur between polymer formation and secretion. Polymannuronan undergoes epimerization at some residues via the action of a periplasmic enzyme. We have shown that another protein, AlgK, forms an isolable complex with the epimerase, and that complex formation requires a third protein AlgX. It is possible that the purpose of the complex is to protect the alginate from degradation by alginate lyase, and to facilitate the delivery of alginate to a porin protein on the outer membrane. During its time in the periplasm, alginate is partially acetylated. Three proteins have been shown to be required for acetylation, AlgI, AlgJ, and AlgF. Their roles remain undefined, and the acetylation process is enigmatic, since there is no acetyl-CoA present in the periplasm. We propose to characterize the protein complexes formed with the epimerase and to determine their influence on its functional properties, and to characterize the acetylation process in molecular detail. We will determine the source of the acetyl groups, and evaluate the roles of the separate proteins in the acetylation process. Kinetics techniques and biophysical methods for characterizing the protein complexes, including gel filtration chromatography, analytical ultracentrifugation and fluorescence anisotropy, will be employed. PUBLIC HEALTH RELEVANCE Pseudomonas aeruginosa is an opportunistic human pathogen that causes life-threatening infections in several different patient populations. A major contributing factor to the virulence of the bacteria and the persistence of their infections in the host is the exopolysaccharide alginate. Biochemical characterization of the steps that lead to formation of mature alginate may provide insights into how to block or ameliorate the effects of infection. [unreadable] [unreadable] [unreadable] [unreadable]